Endotoxin is a negatively charged lipopolysaccharide present in the capsule of Gram-negative bacteria (ref. 3). Endotoxins are complexes of phospholipid (lipid A) and polysaccharide. The endotoxins produced by different bacteria differ in their antigenicity but they all have the same biological effects which are mainly due to lipid A. For the purposes of this description the term endotoxin also comprises enterotoxins. In addition to the negatively charged sugar moieties, an endotoxin contains two phosphate groups which are essential for its toxicity (ref. 3, 4).
Although it is an ubiquitous molecule in the external environment as well as in the gastro-intestinal tract of many species, an endotoxin can be extremely deleterious to these species once it leaves the gastro-intestinal tract e.g causing sepsis and inflammation such as in an abcess even in amounts as low as 10 picogrammes. Yet so far, no important endotoxin detoxifying mechanism has been found in vivo (ref. 5).
Endotoxin is known to induce serious even lethal complications (ref. 5 and 6). In fact, despite the use of antibiotics, this bacterial product is the major cause of death in intensive-care units in Western society.
There are numerous different endotoxins produced by various microorganisms and consequently the actions of endotoxin in vivo are numerous as are the ways it can enter the organism. The symptoms associated with Gram negative infections therefore also vary widely among patients (ref. 7). These symptoms may be further complicated by septic shock of which hypotension, peripheral vasodilation and diffuse intravascular coagulation are the main characteristics (ref. 8). Subsequently organs such as heart (acute heart failure), lungs (adult respiratory distress syndrome). kidney (acute tubular necrosis) and brain may be affected (ref. 8). Endotoxin mediated pathology also comprises the syndrome of multiple organ failure and any other syndrome generally accepted in the art to be directly or indirectly caused by endotoxin.
To date, antibodies directed against endotoxin are the only endotoxin detoxifying proteins known to reduce toxicity irreversibly, but the clinical value of these antibodies remains to be established. Other substances which are able to bind endotoxin, such as lipopolysaccharide binding protein and high density lipoproteins (HDL) (ref. 9), exhibit the major drawback of forming reversible complexes in vivo. Upon dissociation of these complexes, the native (toxic) molecule is produced again. Furthermore although the detoxifying activity of plasma has been noted for some time (ref. 10) efforts to isolate or characterise the substance(s) responsible for this activity have not been successful. Other experimental approaches to treat sepsis include the application of preparations which antagonize the activities of cytokines (e.g. TNF-.alpha.), which are important mediators of endotoxin-induced shock, aggravating the effects of endotoxin in vivo. A major disadvantage of this approach is that these preparations do not detoxify the causative agent but rather block one of the reactions of the body to this toxin. In addition, antagonizing naturally occurring cytokines may cause multiple side effects.
Alkaline phosphatase (EC 3.1.3.1) is a common enzyme present in many species, including man and has been studied extensively. The DNA sequence encoding alkaline phosphatase has even been obtained, but so far no commercial exploitation thereof has occurred. Although the enzyme is routinely applied as antibody label or as a marker for liver and neutrophil function, it's biological relevance is still unknown. Recombinant alkaline phosphatase enzymes with improved specific activity used as indicator reagents are disclosed, e.g. in EP-A-0 441 252. This patent application however mentions nothing regarding anti-endotoxin activity or bone formation of alkaline phosphatase. The cited European patent application describes a number of derivatives in which one amino acid differs from the wild type. The substituents include replacement of Thr 100 by Val or Ile, replacement of Lys 328 by Arg, replacement of Val 99 by Ala, replacement of Thr 107 by Val, replacement of Asp 101 by Ser, replacement of Val 377 by Ala and replacement of Ser 115 by Gly as well as replacement of Ala 103 by Asp. Other derivatives described in the cited patent application are derivatives with M maleimidobenzoyl-N-hydroxysuccinimide ester for carrying out a sandwich EIA and a thiolated mutant of alkaline phosphatase which can be derived through use of succinimidyl-4-N-maleimidomethyl-1-thicapramide cyclohexanecarboxylate. Of all these derivatives no mention is made of the charge carried by the alkaline phosphatase derivative. It is pointed out that all the mentioned derivatives with the exception of the replacement of Ala 103 by Asp have been calculated by us as resulting in a more positive netto charge or an equal netto charge in comparison to the corresponding native alkaline phosphatase.
Alkaline phosphatase is a membrane-bound ecto-enzyme which is known to dephosphorylate extracellular molecules. The enzyme is present in many organs, including intestine, kidney, osteoblasts and neutrophils (ref. 11, 12 and 13). in vitro, it exhibits a pH optimum of approximately 10.5. (ref. 12). This extremely high pH optimum has hampered recognition of its biological relevance (ref. 12-14), because it was felt that this pH level does not occur in biological tissues of the intact organism.
In a number of publications a derivative of alkaline phosphatase and collagen, in particular fibrillar collagen is described. Nothing is mentioned about the netto negative charge of such a derivative, however, we have calculated that the netto charge is positive in comparison to a non-derivatized alkaline phosphatase.
In U.S. Pat. No. 4,409,332 (1983) collagen sutures derivatized with alkaline phosphatase are described as reducing the inflammatory characteristics of collagen. The collagen induced inflammation is not an inflammatory reaction due to endotoxins, it is an inflammation that is generally caused by damage of tissue that has occurred, by the fact that collagen is a heterologous protein which is foreign to the body and by the fact that collagen always induces coagulation in vivo which can subsequently activate inflammatory cells in a number of manners. An inflammation due to infections of the wound is not likely as the authors themselves frequently state that they worked in a sterile environment, using sterile solutions. A person skilled in the art cannot derive from this cited patent publication how alkaline phosphatase coupled to collagen can inhibit the inflammation usually caused by collagen. A number of manners can however be postulated such as, for example by protection of antigens for cells of the specific immunoreaction, thereby prohibiting recognition or by binding positively charged mediators of the non-specific immune reaction as alkaline phosphatase contains negatively charged sugar groups. Another possibility is inhibition of the coagulation cascade by masking collagen or de-phosphorilation of mediators, such as ATP, ADP and platelet activating factor or by binding of positively charged mediators and cofactors.
In the cited document it is stated that even though the hydrolysis functions of outline phosphatase have intensively been studied for more than 50 years no clear image has arisen concerning the value of the enzyme to the organism. In summary the in vivo activity of alkaline phosphatase is not clear. No link is made in the cited document between alkaline phosphatase and bone formation or anti-endotoxin activity. No theoretical background is given to the anti-inflammatory activity of the coupling of alkaline phosphatase to collagen. It is in fact questionable whether a person skilled in the art would even attribute the anti-inflammatory activity to the presence of alkaline phosphatase or whether the fact that specific groups of collagen are protected by the presence of a random derivative provides the anti-inflammatory action. This can be derived from the fact that it is described that the use of cross-linking agent, such as glutaraldehyde or other cross-linking means appears to increase the anti-inflammatory characteristics of the material.
In U.S. Pat. No. 4,394,370 an anti-inflammatory complex of collagen and BMP is described as well as the use thereof in the healing of broken bones, which is further elucidated in U.S. Pat. No. 4,409,332. U.S. Pat. No. 4,394,370 describes the use of reconstituted collagen and dimineralized bone particles or reconstituted collagen and a solubilized bone morphogenetic protein, fabricated in a sponge for in vivo implantation in osseous defects. Both demineralized bone particles and bone morphogenetic protein have demonstrated the ability to induce the formation of osseous tissue in animal and human experiments. Reconstituted collagen conjugate is highly biocompatible and can be fabricated in a variety of configurations. This material can be used as a, grafting implant in plastic and reconstructive surgery, periodontal bone grafting and in endodontic procedures. The structural durability is enhanced by cross-linking with glutaraldehyde, which is also used to sterilize and disinfect the collagen conjugate prior to implantation. It is stated that the soluble factor from demineralized bone, bone morphogenetic protein is osteo inductive and it is also known that the demineralized bone is also condusive to osteogenesis. The use of alkaline phosphatase coupled to BMP and collagen as disclosed in U.S. Pat. No. 4,394,370 is not directed specifically at a bone formation increasing activity of alkaline phosphatase as such but more the fact that alkaline phosphatase linked to collagen has a less inflammatory character than non-derivatized collagen. U.S. Pat. No. 4,394,370 is in particular directed at collagen BMP conjugate sponges and the use of alkaline phosphatase conjugated to such a sponge is merely one of a number of embodiments of BPM-collagen uses and as such is not directed at the same invention as the subject patent application namely the in vivo activity of phosphatase as such or derivatives thereof amongst others for increased bone formation. In U.S. Pat. No. 4,394,370 no mention is made of the anti-endotoxin activity of alkaline phosphatase.
WO 93/00935 describes that the possible role of the enzyme alkaline phosphatase in promoting the calcification of bone has been postulated for many years. However that the relevance of such in vitro mineralization studies to the situation in vivo has been questioned, particularly in view of the relatively high concentrations of phosphate esters used in the in vitro studies and also because the rate of hydrolysis of the phosphate esters and physiological pH levels would be expected to be too low to be relevant to the process of mineralization. In the cited patent application Beertsen et al. describe that combination of a biocompatible carrier material, preferably one which can mineralize to some degree itself, such as fibrillar collagen with a quantity of a phosphatase enzyme will promote mineralization. Preferably the combination of alkaline phosphatase with the carrier is brought about by incubating the carrier with the enzyme in the presence of a coupling agent capable of covalently bonding with the carrier and with the enzyme. Suitable coupling agents are described as biotinavidin, glutaraldehyde and 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide HCl. A particularly preferred coupling agent is known as succinimidyl-s-acetyl-thioacetate (SATA) in combination with maleimido hexanoyl-N-hydroxysuccinimide (MHS) wherein the carrier is incubated with SATA and the enzyme with the MHS. The products of these two incubation processes are combined and allowed to react to produce an implant material. The cited document describes that the coupling of alkaline phosphatase to collagen improves the osteogenesis when such a complex is placed in situ of the wound. The alkaline phosphatase is used in combination with a product already known to stimulate bone formation. No description is given of use of alkaline phosphatase as such or as a derivative with a particular altered charge. It is pointed out that a derivative of alkaline phosphatase with fibrillar collagen has an increased positive charge in comparison to non-derivatized alkaline phosphatase. A derivative of alkaline phosphatase with fibrillar collagen is not suitable for systemic application as fibrillar collagen induces intravascular platelet activation leading to embolisms. Therefore, a complex of fibrillar collagen and alkaline phosphatase could not be used in a method for treating osteoporosis or osteomalacia or any other bone defect which requires systemic application. It can only be used when immobilized in situ at the location of a wound.